1. Field of Invention
The present invention relates to a method for measuring, rapidly and in a simplified manner, the number of living microorganisms in a sample, and a measuring kit, which can be utilized in a wide variety of fields including metal working, paints and foodstuffs in which a problem of spoilage will arise, and in diagnostics for detecting bacteria in urine.
2. Background Information
Water-soluble metal working fluids such as cutting fluids, rolling coolants or polymer quenchants, liquid foodstuffs such as liquid flavoring matters, liquid foods and drinks or alcohols, water-soluble paints, and the like are used under conditions suitable for growth of microorganisms. Thus, depending on the degree of administration, such fluids are sometimes spoiled as a result of considerable growth of microorganisms.
In order to prevent spoilage of such water-soluble metal working fluids or liquid foodstuffs at an earlier stage and to keep them in a desirable state, it is necessary to know the exact number of microorganisms, particularly living aerobic bacteria in the sample.
For measurement of the number of living aerobic microorganisms such as yeasts and bacteria in various specimens, there has heretofore been known a method, for example, in which a predetermined amount of specimen is cultivated on an agar medium, the number of colonies formed are counted, and from the number of colonies, the number of living microorganisms is calculated.
This method, however, has disadvantages in that special equipment, such as an incubator, is needed, and a long time, usually 48 hours, is needed until the results of measurement are obtained.
Thus, for rapid measurement of living aerobic microorganisms, a method in which enzyme activity (catalase activity) is measured (Japanese Patent Application Laid-Open No. 74095/1982), and a method in which microorganisms are dyed with fluorescent coloring matter (Japanese Patent Application Laid-Open No. 138185/1987) were proposed.
However the former method has a problem that hydrogen peroxide has to be used, which is unstable. For the latter method, special equipment, such as a fluorescent photometer, is needed and, therefore, the method can be employed only in specified conditions.
Thus there was proposed a method in which microorganisms are adsorbed on a negatively charged filter and then dyed with coloring matter (Japanese Patent Application Laid-Open No. 124767/1989).
This method, however, has various disadvantages. For example, in the adsorption of microorganisms on the filter, pretreatment with a strong acid of pH 1-3 is needed, and the operation is very complicated; for example, caution should be taken to prevent the strong acid, such as hydrochloric acid, from coming into contact with the skin of an operator, and for removal of excessive free coloring matter, washing should be carried out at least twice.
In addition, U.S. Pat. No. 4,336,337 discloses an apparatus in which urine, for example, is dyed with safranine in the presence of EDTA and adsorbed on a positively charged filter and, thereafter, after washing with a cleaner of pH 2.7-4.0, the number of living microorganisms is determined according to the intensity of color.
In this case, however, a vacuum pump or equipment for suction is needed for removal of excessive coloring matter, and thus the size of the apparatus becomes large and the place where the apparatus is to be located, is limited.